POS1428 AMLEXANOX INHIBITS PRODUCTION OF TYPE I INTERFERON AND SUPPRESSES B CELL DIFFERENTIATION AND IMMUNOGLOBULIN PRODUCTION IN VITRO: A POTENTIAL NOVEL THERAPEUTIC OPTION FOR SLE

Abstract

BackgroundThe immunopathology of systemic lupus erythematosus (SLE) is characterized by production of autoantibodies and aberrant activation of the type I interferon (IFN) system. In SLE, type I IFNs directly and indirectly affect differentiation and survival of autoreactive B cells, partly by IFN induced release of B cell activating factor (BAFF). TANK-binding kinase 1 (TBK-1) is an important upstream mediator of type I IFN production, which has been implicated in several systemic autoimmune diseases [1]. Additionally, B cell intrinsic TBK-1 was recently shown to be essential for germinal center (GC) formation and proper B cell development in mice [2]. Amlexanox (AMX), a small molecule TBK-1 inhibitor, has been used to treat aphthous ulcers and asthma. Systemic administration of AMX has indicated that the drug is well tolerated with little evidence of safety concerns [3]. Therefore, we hypothesize that AMX, by inhibition of TBK-1, could be a novel therapeutic option in SLE.ObjectivesTo determine whether AMX can inhibit type I IFN and IFN induced BAFF production, and to unravel its potential direct effects on B cells in human in vitro culture systems.MethodsCultured PBMC from healthy donors were stimulated with Imiquimod, CpG-A, Poly:IC, G3-YsD or 3p-hpRNA to induce type I IFN production through various routes, with or without presence of AMX. Production of type I IFN was assessed by a HEK IFN-α/β reporter cell line and the ability of supernatants to induce transcription of the BAFF and MX1 genes was measured by real-time quantitative PCR. Sorted CD19+ B cells were cultured for 6 or 9 days under GC-like conditions with CD40L and IL-21 (4), as well as IFN, BAFF, and AMX at various concentrations. Viability and differentiation into CD38highCD27highCD138+/- cells was assessed by spectral flow cytometry. Immunoglobulin M and IgG were measured in supernatants by ELISA.ResultsAmlexanox significantly inhibited production of type I IFN through all investigated endosomal and cytosolic routes (p=0.03 to p=0.001). Likewise, supernatants from cells treated with AMX were unable to induce expression of BAFF and MX1. The inhibiting capacity of AMX in this setting was comparable to blocking of the interferon alpha/beta receptor (IFNAR) with a monoclonal antibody. As could be expected, AMX did not affect expression of BAFF and MX1 upon IFNAR ligation, indicating no effect on signaling downstream of the receptor. To mimic an autoimmune setting, IFN and BAFF was added to the CD40L/IL-21 B cell culture system, resulting in increased B cell differentiation. Addition of AMX resulted in significantly decreased differentiation into CD38highCD27highCD138+/- cells (p<0.0001) without affecting cell viability. Correspondingly, production of IgM and IgG was lower in the AMX condition.ConclusionOur data demonstratein vitroinhibitory effects of AMX on type I IFN production and B cell differentiation in primary human cells. Inhibition of TBK-1 is a promising therapeutic target for treatment of SLE warranting further investigations.